The global analysis of the biochemical activities of proteins and their modifications is in a nascent stage. In the previous funding period we have developed technologies for producing large numbers of active proteins and displaying them in a high density protein microarray format. A first generation proteome array collection was built and screened for a variety of interactions and activities. In this application we propose to improve the yeast protein chip content and technology and apply it to the study of protein phosphorylation. We will complete the cloning of all yeast ORFS in the versatile GATEWAY cloning system and construct a high quality expression collection that produces the proteins with tags at their carboxy termini. The proteins will be overexpressed and used to construct a comprehensive yeast proteome array. We will attempt to establish quantitative methods for probing protein arrays and compare proteome chip technology with two hybrid and affinity purification methods for detecting protein-protein interactions. The comprehensive array and expression collection will be used to investigate protein phosphorylation on a large scale. The array will be used to identify proteins that interact with protein kinases. An in vitro phosphorylation map will be generated by incubation of each yeast protein kinase with the proteome chip in the presence of 33P-gamma-ATP. A consensus phosphorylation site will be determined for each kinase, and mass spectrometry will be used to map the in vitro and in vivo sites of phosphorylation on many yeast substrates. A subset of kinase-substrate interactions will be validated in vivo using mutant strains. Information from this study will be combined with public information to generate a phosphorylation map for yeast. The information and reagents from this work will be made available to the entire scientific community. The technology developed and information gathered are expected to be applicable to multicellular organisms.